A Recombinant Newcastle Disease Virus with Low-Level V Protein Expression Is Immunogenic and Lacks Pathogenicity for Chicken Embryos

Abstract

Newcastle disease virus (NDV) edits its P-gene mRNA by inserting a nontemplated G residue(s) at a conserved editing site (3-UUUUUCCC-template strand). In the wild-type virus, three amino-coterminal P-gene-derived proteins, P, V, and W, are produced at frequencies of approximately 68, 29, and 2%, respectively. By applying the reverse genetics technique, editing-defective mutants were generated in cell culture. Compared to the wild-type virus, mutants lacking either six nucleotides of the conserved editing site or the unique C-terminal part of the V protein produced as much as 5,000-fold fewer infectious progeny in vitro or 200,000-fold fewer in 6-day-old embryonated chicken eggs. In addition, both mutants were unable to propagate in 9-to 11-day-old embryonated specific-pathogen-free (SPF) chicken eggs. In contrast, a mutant (NDV-P1) with one nucleotide substitution (UUCUUCCC) grew in eggs, albeit with a 100-fold-lower infectious titer than the parent virus. The modification in the first two mutants described above led to complete abolition of V expression, whereas in NDV-P1 the editing frequency was reduced to less than 2%, and as a result, V was expressed at a 20-fold-lower level. NDV-P1 showed markedly attenuated pathogenicity for SPF chicken embryos, unlike currently available ND vaccine strains. These findings indicate that the V protein of NDV has a dual function, playing a direct role in virus replication as well as serving as a virulence factor. Administration of NDV-P1 to 18-day-old embryonated chicken eggs hardly affected hatchability. Hatched chickens developed high levels of NDV-specific antibodies and were fully protected against lethal challenge, demonstrating the potential use of editing-defective recombinant NDV as a safe embryo vaccine.Newcastle disease virus (NDV) belongs to the genus Rubulavirus within the family Paramyxoviridae. Recent findings, however, have indicated that NDV is only distantly related to other members of the genus Rubulavirus, and it has been suggested that NDV should be assigned to a new genus within the subfamily Paramyxovirinae (6). NDV isolates are further categorized based on pathogenicity for chickens into velogenic, mesogenic, and lentogenic strains corresponding to high-, moderate-, and low-virulence strains, respectively. The molecular basis for this distinction lies mainly in the amino acid sequence of the protease cleavage site of the fusion (F) protein (14, 25). The precursor fusion glycoprotein F0 has to be cleaved into F1 and F2 for the progeny virus to be infectious and to be able to undergo multiple rounds of replication. Recently, experimental evidence for the presence of a direct correlation between the sequence of the cleavage site and NDV virulence was provided by changing the protease cleavage site of a lentogenic strain of NDV (GGRQGR7 L) into the consensus cleavage site of a velogenic strain (GRRQRR7 F). A dramatic increase in virulence of the genetically modified virus indicated that the key determinant for NDV virulence is the cleavage efficiency of t...

Contribution of the length of the HN protein and the sequence of the F protein cleavage site to Newcastle disease virus pathogenicity

“…This suggests that the F protein cleavage site is not the sole determinant of NDV virulence but that other mutations in the NDV genome may cause the increase in pathogenicity. Recently, we have shown that the NDV V protein, which is produced as a result of editing of the P gene mRNA, plays a role in pathogenicity (Mebatsion et al, 2001). It was demonstrated that the level of V protein expression correlates directly with NDV virulence for chicken embryos.…”

“…Introductions of F and HN sequence alterations were done using the USE Mutagenesis kit (Amersham Pharmacia). Mutagenesis of the F cleavage site to generate rNDVF1 was done as described previously (Mebatsion et al, 2001). To alter the position of the HN stop codon, PCR was performed on pfl-NDV-1 using the forward primer P3F and the reverse primer P3R (Table 1).…”

Review of Newcastle disease virus with particular references to immunity and vaccination

“…Reverse genetics can also be used to produce ND vaccines with reduced pathogenicity for chicken embryos. One such virus expresses low levels of the V protein, exhibits impaired replication, but still induces protective antibody levels in hatched chickens (Mebatsion et al, 2001).…”

Molecular study of the quasispecies evolution of a typical pigeon paramyxovirus type 1 after serial passages in pigeons by contact

“…The matrix protein (M) is involved in the budding process of the newly formed virus particles (Peeples et al , 1992), while the haemagglutinin-neuraminidase (HN) and fusion protein (F), two glycoproteins anchored in the virion envelope, are involved in the binding and/or fusion to the host cell membrane, and, for HN, in the release of the newly formed virus particles (Yusoff & Tan, 2001). The P gene also encodes two non-structural proteins, called V and W, which seem to be involved in the pathogenesis of the virus (Steward et al , 1995;Mebatsion et al , 2001;Park et al , 2003).…”